Project 4 - Protein C Translational Studies. Project Leader: John H. Griffin Ph.D. The PROWESS phase III clinical trial showed that activated protein C (APC) reduced mortality where neither purely anticoagulant nor purely anti-inflammatory agents were effective. Thus, APC infused into humans, as in animals, exerts life-saving activities in addition to its anticoagulant actions. Recent in vitro and in vivo studies support a new paradigm in which APC exerts protective anti-inflammatory and anti- apoptotic direct effects on cells via mechanisms involving activation of Protease Activated Receptor1 (PAR1) by APC bound to Endothelial Protein C Receptor (EPCR). APC's anti-inflammatory and anti- apoptotic in vivo activities may result from its ability to alter gene expression profiles. Both inflammation and apoptosis contribute to the pathogenesis of thrombosis. Thus, recombinant (r) APC is promising for treating thrombotic disorders. We will prepare numerous murine APC mutants and study them as potential therapeutic agents using murine models to define their in vivo activities. APC's anticoagulant activity and APC's direct effects on cells in vitro will be determined in studies of the EPCR-dependent, PAR1-dependent abilities of APC to inhibit apoptosis and to alter endothelial cell gene expression. To assess APC's in vivo activities, we will use murine models and determine for wild type and mutant APC's the following in vivo properties: 1) antithrombotic activity; 2) anti-inflammatory activity; and 3) alteration of gene expression in the absence of injury. In preliminary work, we identified two APC mutants deficient in anticoagulant activity with normal in vitro anti-apoptotic activity. Such APC variants may provide APC's beneficial EPCR-dependent, PAR 1-dependent direct effects on cells with reduced risk of serious bleeding. Because APC protectively targets both clotting factors and cell surface receptors, the results of this project may lead directly to development of novel, multi-functional agents for treatment of a variety of thrombotic disorders.